Electrochemical Signal-Off Competitive Immunoassay of Chromogranin A toward a Sandwiched Graphene Oxide Structure for Neuroendocrine Tumor Detection

Abstract

Neuroendocrine tumors (NETs), often misdiagnosed and mistreated, require early detection for precise therapeutic interventions. This study presents a newly developed competitive electrochemical immunosensor for sensitive and selective detection of chromogranin A (CgA), a key biomarker for diagnosing and monitoring NETs. The sensor, featuring a sandwiched structure with versatile and multifunctional graphene oxide (GO), utilizes polyethylenimine-capped gold nanoparticles (PEI-AuNPs) to enhance the electroreactivity and biocompatibility of a screen-printed electrode (SPE). The immunosensor operates by immobilizing standard CgA antigens on the PEI-AuNPs/GO-modified SPE surface and employing GO nanotags loaded with anti-CgA antibodies (Ab) and ferrocene monocarboxylic acid (Fc) redox probes to capture target CgA. As the CgA concentration increases, the current response of the immunosensor decreases due to a reduction in the amount of Fc/Ab/GO tags on the electrode surface. This reduction occurs because the nanotags bind to the free CgA in the sample rather than the CgA immobilized on the electrode. The immunosensor demonstrates a good linearity (0.10-50 ng mL^-1), a low detection limit of 90 pg mL^-1, and high accuracy in detecting CgA levels in human serum samples. With its high specificity, long-term stability, and excellent reproducibility, our cost-effective and user-friendly platform holds promise for clinical screening and point-of-care diagnosis of NETs. Further optimization of the immunosensor's design and exploration of its use for additional biomarkers could enhance NETs' diagnosis and provide advancements in managing other related health conditions.

Keywords: chromogranin A; competitive assay; electrochemical immunosensor; neuroendocrine tumor; sandwich graphene oxide architecture.

1. Schematic Illustration of Fabrication and CgA Detection of Competitive Electrochemical Immunosensor

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SEM images of (A) unmodified SPE and SPEs modified with (B) PEI-AuNPs, (C) GO, and (D) PEI-AuNPs/GO.

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Optimization of (A) PEI-AuNPs concentration, (B) CgA concentration, (C) incubation time for immobilization of CgA, (D) incubation time for binding of tags to CgA-modified sensing surface after competitive capture, (E) incubation time for adsorption of anti-CgA on Fc/GO tags to form anti-CgA/Fc/GO tags, and (F) incubation time for immunoreaction time between anti-CgA on Fc/GO tags and CgA in 1.0 ng mL^–1 CgA solution during competitive capture.

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DPV sensorgrams of the immunosensors after being incubated with anti-CgA/Fc/GO solution left from competitive capture of CgA in different CgA concentrations in (A) PBS and (B) 50-fold diluted human serum, and (C) comparison of two calibration curves based on changes of the DPV peak current versus the logarithm of the CgA concentration.

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(A) Interference study of immunosensor for detection of CgA in blank, 100 ng mL^–1 interferences solutions and their mixture, and 1.0 ng mL^–1 CgA solutions (with and without the presence of 100 ng mL^–1 interferences or mixture), (B) immunosensor’s reproducibility for detection of CgA in blank and 1.0 ng mL^–1 CgA solution, (C) immunosensor’s repeatability for detecting CgA in 1.0 ng mL^–1 CgA solution, and (D) stability of the developed immunosensor in the detection of 1.0 ng mL^–1 CgA.